Aging is associated with a loss of ability to modulate responses to physiological stress. While the mechanisms underlying these age-related alterations are unclear, evidence implicates increased generation of reactive oxygen species (ROS) and associated oxidative stress. We have demonstrated that senescence is associated with reduced stress tolerance. Our pilot experiments suggest that heat stress produces augmented radical generation, impaired antioxidant enzyme (AE) profiles, and widespread cellular injury in older animals. The goal of this proposal is to study the mechanisms of ROS formation and oxidative injury at cellular and subcellular levels to determining how the aged organism copes with physiological stress. We will also modulate the stress response by manipulating redox status in both an in vivo animal model and an in vitro primary cell culture system. The guiding hypothesis for this research program is that aging organisms have a reduced ability to cope with physiological stress due to an exaggerated production of ROS and concomitant oxidative damage. We will examine this hypothesis by: (1) determining the functional relationship between ROS generation, AE responsiveness, and cellular injury in selected tissues in senescent compared to young rats following heat stress; (2) determining whether the age-related differences in ROS generation, AE responsiveness, and cellular injury following heat stress are due to alterations at the cellular level; (3) determining whether physiological stress produces age-related alterations in transcription factor activation and, if so, whether these alterations are part of the mechanism contributing to oxidative damage; (4) manipulating cellular redox status in young and old animals before the application of heat stress. We will use a unique integrated approach that includes whole animal, cellular, molecular, and novel in vivo gene transfer techniques involving AE overexpression to pursue basic mechanisms in the stress response. By using state-of-the-art techniques, we will be able to address important mechanistic questions involving ROS generation, oxidative injury, and aging that will have widespread application to numerous clinical problems (cancer, cardiovascular disease, septic shock, frailty) in aged populations. The results of this research will allow us to design new therapies to protect the elderly against situations involving physiological stress, and potentially, many diseases associated with aging.